2000 characterization of the coronavirus mouse hepatitis virus strain a59 small membrane protein e

JOURNAL OF VIROLOGY,0022-538X/00/$04.0010

Mar. 2000, p. 2333–2342 Vol. 74, No. 5

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Characterization of the Coronavirus Mouse Hepatitis VirusStrain A59 Small Membrane Protein E

MARTIN J. B. RAAMSMAN,1 JACOMINE KRIJNSE LOCKER,2 ALPHONS DE HOOGE,1

ANTOINE A. F. DE VRIES,1 GARETH GRIFFITHS,2 HARRY VENNEMA,1 AND PETER J. M. ROTTIER1*

Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Institute of Virology,and Institute of Biomembranes, Utrecht University, 3584 CL Utrecht, The Netherlands,1

and European Molecular Biology Laboratory, Heidelberg, Germany2

Received 23 June 1999/Accepted 2 December 1999

The small envelope (E) protein has recently been shown to play an essential role in the assembly ofcoronaviruses. Expression studies revealed that for formation of the viral envelope, actually only the E proteinand the membrane (M) protein are required. Since little is known about this generally low-abundance virioncomponent, we have characterized the E protein of mouse hepatitis virus strain A59 (MHV-A59), an 83-residuepolypeptide. Using an antiserum to the hydrophilic carboxy terminus of this otherwise hydrophobic protein, wefound that the E protein was synthesized in infected cells with similar kinetics as the other viral structuralproteins. The protein appeared to be quite stable both during infection and when expressed individually usinga vaccinia virus expression system. Consistent with the lack of a predicted cleavage site, the protein was foundto become integrated in membranes without involvement of a cleaved signal peptide, nor were any othermodifications of the polypeptide observed. Immunofluorescence analysis of cells expressing the E proteindemonstrated that the hydrophilic tail is exposed on the cytoplasmic side. Accordingly, this domain of theprotein could not be detected on the outside of virions but appeared to be inside, where it was protected fromproteolytic degradation. The results lead to a topological model in which the polypeptide is buried within themembrane, spanning the lipid bilayer once, possibly twice, and exposing only its carboxy-terminal domain.Finally, electron microscopic studies demonstrated that expression of the E protein in cells induced theformation of characteristic membrane structures also observed in MHV-A59-infected cells, apparently con-sisting of masses of tubular, smooth, convoluted membranes. As judged by their colabeling with antibodies toE and to Rab-1, a marker for the intermediate compartment and endoplasmic reticulum, the E proteinaccumulates in and induces curvature into these pre-Golgi membranes where coronaviruses have been shownearlier to assemble by budding.

Coronaviruses, a family of viruses belonging to the newlyestablished order of the Nidovirales (for reviews, see references8 and 37) have enveloped virions containing a nonsegmented,plus-stranded RNA genome. The RNA is packaged by thenucleocapsid (N) protein into a helical nucleocapsid. The sur-rounding envelope contains three, and sometimes four, mem-brane proteins. The spike (S) protein, a type I glycoprotein,occurs as trimers that constitute the characteristic surface pro-jections. These function primarily in virus entry, being respon-sible for binding to the receptor on the target cell and formediating fusion of viral and cellular membranes. The mem-brane (M) protein is a triple-spanning glycoprotein. It is themost abundant envelope protein component having essentialfunctions in virus assembly. The hemagglutinin-esterase pro-tein is present in only a subset of coronaviruses. The type Iglycoprotein occurs in virions in disulfide-linked homodimericform. Its biological role in the virus life cycle has not been wellestablished.

The small envelope (E) protein was only recently recognizedas a structural component of the coronavirion (12, 26, 48, 49).Although very little is still known about its features, the Eprotein appears to be surprisingly important for assembly ofthe viral envelope. By coexpression of the genes encoding themouse hepatitis virus strain A59 (MHV-A59) membrane pro-

teins we showed that virus-like particles (VLPs) morphologi-cally mimicking normal virions were produced only when the Eprotein was present, while the S protein was dispensable (48).Similar particles were observed after coexpression of transmis-sible gastroenteritis virus (TGEV) membrane proteins (1a).

Coronavirus E proteins vary in size from about 76 to 109amino acids (for a review, see reference 38). Consistent withtheir membrane association (12, 40, 48, 49), the proteins aregenerally quite hydrophobic in nature, particularly in theirN-terminal half (see Fig. 1). The MHV-A59 E protein wasreported to be acylated on the basis of a biochemical assay(49), but attempts to directly label the TGEV protein withpalmitic acid failed (12). By immunofluorescence, the E pro-tein was observed in infected cells in a granular (IBV) (40) orpunctate (BCV and MHV-A59) (1, 49) pattern, as well as atthe plasma membrane (12, 40, 49). The cell surface stainingwith a C-terminus-specific antibody led Godet et al. (12) tosuggest a CexoNendo membrane topology for the TGEV Eprotein, i.e., with its C and N termini exposed luminally andcytoplasmically, respectively.

Very recently Fischer et al. (10) described the effects ofmutations in the E protein of MHV-A59. Using clusteredcharged-to-alanine mutagenesis and targeted RNA recombi-nation, two mutant viruses were obtained that were partiallytemperature sensitive, forming small plaques at the nonper-missive temperature, and markedly thermolabile when grownat the permissive temperature. Most interestingly, one of theseviruses appeared to have strikingly aberrant morphology whenviewed by electron microscopy. Many virions showed pinched

* Corresponding author. Mailing address: Institute of Virology, Fac-ulty of Veterinary Medicine, Utrecht University, P.O. Box 80.165, 3508TD Utrecht, The Netherlands. Phone: 31-30-2532462. Fax: 31-30-2536723. E-mail: [email protected].

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and elongated shapes, which is consistent with a role for the Eprotein in particle morphogenesis.

In order to obtain more insight into the function of the Eprotein in coronavirus infection, particularly regarding its rolein viral assembly, we have analyzed a number of its basicfeatures. We have studied, in addition to its appearance andfate in infected cells, its independent properties by expression,as well as its topology in cellular and viral membranes.

MATERIALS AND METHODS

Cells and viruses. Mouse L cells, OST7-1 cells (9), and RK13 cells weremaintained in Dulbecco modified Eagle medium (DMEM) containing 10%heat-inactivated fetal calf serum (FCS)–100 IU of penicillin per ml–100 mg ofstreptomycin per ml (DMEM–10% FCS), supplemented in the case of OST7-1cells with 400 mg of G-418 (Geneticin; Gibco BRL) per ml. BHK-21 cells andCEF cells were maintained in Glasgow minimal essential medium (MEM) con-taining the same additional ingredients.

MHV-A59 was propagated in Sac(2) cells as described previously (42). Therecombinant vaccinia viruses vTF7-3 and MVA-T7 expressing bacteriophage T7RNA polymerase were propagated in RK13 and CEF cells, respectively, asdescribed previously (11, 43).

Expression vectors. The expression construct pT7Ts-E carrying the MHV-A59E gene behind a T7 promoter was made by using the vector pT7Ts (a kind giftof P. Krieg). The E gene was obtained as a PCR fragment amplified from cDNAclone pRG86 (2) by using primer 471 (59-dCACGCAGCTCGAAACATATGTTTA-39) and primer 496 (59-dGGATTAGATATCATCCACCTCTA-39). It wascloned into pNoTa (5 Prime33 Prime, Inc.). From the resulting plasmid pNoTa/T7-5b, the E gene was subsequently retrieved with NdeI and BamHI, and thisfragment was treated with Klenow DNA polymerase. pT7Ts was first digestedwith EcoRI and XbaI and then treated with Klenow enzyme and religated. The

plasmid was then cut with SpeI and treated again with Klenow DNA polymerase,and finally the E gene fragment was ligated into it to yield pT7Ts-E.

The expression plasmid pTUM-M containing the MHV-A59 M gene behind aT7 promoter has been described elsewhere (28), as has pAVI02, a pBluescriptKS(1) construct carrying the equine arteritis virus (EAV) Gs gene (6).

Antibodies. To obtain a serum against the MHV-A59 E protein, we con-structed a plasmid encoding an N-terminally His-tagged fusion protein of themajor part of the EAV Gs protein ectodomain linked to the C-terminal 34-residues sequence of the E protein (Fig. 1B). To this end, we amplified therelevant region of the plasmid pNoTA/T7-5b by PCR using the primers 485(59-dGGAATTCTTTGGTGCTGTCCCCTTC-39; nucleotides 15554 to 15571 ofthe MHV-A59 RNA sequence) and the M13 reverse sequence primer (Pharma-cia). This PCR product was digested with EcoRI and HindIII and cloned intopBluescript SK(2) to generate pBM.cE. To obtain the fragment encoding the161-residues Gs polypeptide, we performed a PCR with the primers 483 (59-dCGGGATCCATCGCCCGCAGCTTGG-39, nucleotides 9897 to 9914 of the EAVRNA sequence) and primer 484 (59-dGGAATTCAACACTTCCACAGATGAAGC-39; genomic positions 10362 to 10382). This fragment was cloned as aBamHI/EcoRI fragment into pBluescript SK(2) to generate pBE.GS-ecto. Thenthe EcoRI/HindIII fragment from pBM.cE was cloned into pBE.GS-ecto behindthe EAV fragment which resulted in the unintentional insertion of two codons atthe Gs-E junction site adding two amino acids (Asn and Ser). The chimeric Gs-Efragment was cloned as a BamHI fragment into the His-tag vector pQE10(Qiagen) yielding pGSectoEendo. The protein was expressed in Escherichia coli,isolated, and purified on a Ni-nitriloacetic acid column (Invitrogen) according tothe manufacturer’s instructions. Antibodies were elicited by subcutaneous injec-tion of the fusion protein (75 mg in Freund complete adjuvant) into a rabbit fromwhich blood had been taken the day before to obtain a preserum (designatedpreE). The rabbit was boosted similarly after 3, 7, 11, and 15 weeks using 250 mgof protein in Freund incomplete adjuvant each time. Animals were bled 17 weeksafter the first immunization.

The monoclonal antibody J1.3 (a kind gift of J. Flemming) recognizes the

FIG. 1. (A) Amino acid sequence of the MHV-A59 E protein and its hydropathy profile as determined by Kyte and Doolittle (23) with a 8-residue moving window.Peaks extending upwards indicate hydrophobic domains; those pointing downwards represent hydrophilic regions. (B) Structure of the fusion protein for antibodypreparation against E protein. A construct was prepared consisting of the ectodomain of the EAV GS protein (residues 23 to 184) and the MHV-A59 E protein(residues 49 to 83, underlined in panel A) preceded by a 6-histidine stretch.

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N-terminal domain of the MHV-A59 M protein (designated aMN), as has beendescribed (5). Also described previously was a rabbit serum against a peptidecorresponding to the M protein’s C terminus (aMC [19]), as well as a polyclonalrabbit serum against MHV virions (aMHV [32]).

Infection, transfection, and metabolic labeling. Subconfluent monolayers ofOST7-1 cells grown in 10-cm2 dishes were washed with phosphate-buffered saline(PBS) containing 50 mg of DEAE-dextran per ml (PBS-DEAE) and 1% of FCSand inoculated using this medium with MHV-A59 at a multiplicity of infection(MOI) of 10 to 50 for 60 min at 37°C.

For expression of the MHV-A59 M and E genes and of the EAV Gs gene,subconfluent monolayers of OST7-1 or BHK-21 cells in 10-cm2 dishes werewashed with DMEM and inoculated with vTF7-3 or MVA-T7 at an MOI of 10in DMEM for 45 min at 37°C. Cells were then washed with DMEM and trans-fected with the plasmid of choice. For this purpose, 200 ml of DMEM containing10 ml of Lipofectin reagent (Life Technologies, Inc.) was mixed with 5 mg ofplasmid DNA and added to the cells. After a 10-min incubation at room tem-perature, 800 ml of DMEM was added, and the cells were incubated furtherinitially at 37°C for 1 h and subsequently at 32°C for the indicated times; in thecase of MVA-T7-driven expression the cells were kept at 37°C at all times.

For radioactive labeling of proteins, MHV-A59-infected cells and cells ex-pressing genes from plasmids were starved for 30 min by incubation with MEMlacking methionine and cysteine (GIBCO) and supplemented with 10 mMHEPES (pH 7.4). Cells were labeled with 80 mCi of 35S In Vitro Labeling Mix(Amersham) in the same medium for the times indicated. For pulse-chase ex-periments, the cells were washed twice after the labeling with prewarmed chasemedium (DMEM–10% FCS, 2 mM methionine and cysteine, and 10 mMHEPES [pH 7.4]) and incubated further in the same medium.

Immunoprecipitation, immunoisolation, and protein analysis. After labeling,cells were washed once with PBS containing 50 mM Ca21 and 50 mM Mg21.They were then lysed on ice in 1 ml of lysis buffer (20 mM Tris, pH 7.6; 150 mMNaCl; 1% Nonidet P-40 [NP-40]; 0.5% deoxycholate; and 0.1% sodium dodecylsulfate [SDS]) per 10-cm2 dish. The lysates were cleared by centrifugation for 15min at 13,000 rpm at 4°C in an Eppendorf centrifuge. To analyze proteinspresent in the culture media, cell supernatants were taken off, cleared by cen-trifugation for 15 min at 4°C and 4,000 rpm, and then mixed with a one-fifthvolume of a 53 concentrated lysis buffer: 100 mM Tris (pH 7.6), 150 mM NaCl,5% (vol/vol) NP-40, 2.5% (wt/vol) deoxycholate (DOC), 0.5% (wt/vol) SDS. Toanalyze proteins present in cells and culture media together, as was done in theexperiments of Fig. 2 and 3, total lysates were prepared by adding a one-fifthvolume of 53 concentrated lysis buffer to the culture media. The combinedlysates of cells and media were cleared by centrifugation as described above forconventional cell lysates.

For immunoprecipitation of proteins, 100- or 150-ml aliquots of lysates werediluted to 1 ml with immunoprecipitation buffer (IP buffer; 20 mM Tris [pH 7.6],150 mM NaCl, 5 mM EDTA, 0.5% [vol/vol] NP-40, 0.1% [wt/vol] DOC, 0.1%[wt/vol] SDS), and antibodies (3 ml of rabbit sera and 100 ml of monoclonalantibody tissue culture supernatant) were added. The solutions were incubatedat 4°C for at least 3 h, after which 30 ml of Pansorbin (Calbiochem) was added,and the incubation continued for at least 1 h. Immune complexes were thencollected by centrifugation, washed three times with wash buffer I (20 mM Tris[pH 7.6], 150 mM NaCl, 5 mM EDTA, 0.1% NP-40) and once with wash bufferII (20 mM Tris [pH 7.6], 0.1% NP-40).

Immunoisolation of virus particles was performed by diluting 450 ml of clearedcell culture supernatant with a similar volume of TNE (20 mM Tris [pH 7.6], 50mM NaCl, 1 mM EDTA) containing antibodies (3 ml of rabbit serum or 100 mlof monoclonal antibodies). The solution was incubated overnight at 4°C, afterwhich 30 ml of Pansorbin was added to each sample. Incubation was continuedfor 1 h, after which immune complexes were collected by centrifugation andwashed three times with TNE.

For analysis of the proteins, the washed immune complexes were resuspendedin 30 ml of Laemmli sample buffer containing 50 mM dithiothreitol (DTT) andanalyzed by electrophoresis in SDS-polyacrylamide gels (PAG). The gels werefixed in 10% acetic acid–50% methanol for at least 30 min and incubated foranother 30 min in 1 M sodium salicylate. Finally, the gels were dried andsubjected to fluorography at 280°C. Radioactivity in protein bands in PAG wasquantitated by using a PhosphorImager and ImageQuant (Molecular Dynamics)analysis.

Proteinase K treatment of MHV virions. After labeling of MHV-A59-infectedOST7-1 cells, the culture medium was collected, cleared, and adjusted to 0.01 MTris (pH 7.8) and 1 mM CaCl2 (proteinase K reaction buffer) by using a 1003concentrated stock solution. Proteinase K was then added from a stock solutionto some samples to a concentration of 150 mg/ml, and all samples were incubatedfor 1 h on ice. The enzymatic treatment was stopped by adding phenylmethyl-sulfonyl fluoride (PMSF) to a concentration of 7.5 mM while keeping the sam-ples on ice. A one-fifth volume of concentrated lysis buffer was then added toeach sample, and immunoprecipitations were performed as detailed above but inthe presence of 5 mM PMSF in all solutions.

In vitro transcription and translation. In vitro transcription reactions werecarried out by using T7 RNA polymerase (Boehringer Mannheim) according tothe manufacturer’s instructions in 50-ml volumes containing 4 mg of PstI-linear-ized pT7Ts-E. After a 1-h incubation at 37°C, the RNA was purified by chro-

matography in a Sephadex G50 column and precipitated with ethanol, and thedried pellet was dissolved in 50 ml of TNE.

Translations of the mRNAs (0.8 ml of RNA transcript in a 10-ml reaction) weredone for 1 h at 30°C in the Promega rabbit reticulocyte lysate system in thepresence or absence of canine microsomal membranes (Promega). The sampleswere then split and diluted to 1 ml with IP buffer. Antisera (3 ml of the rabbitpreE serum or the anti-E serum) were added, and the normal immunoprecipi-tation procedure was followed.

Immunofluorescence analysis of protein membrane topology. BHK-21 cellsexpressing the MHV-A59 E or M protein were surface permeabilized by usingStreptolysin O (SLO; purchased from S. Bhakdi, Johannes Gutenberg-Univer-sitat, Mainz, Germany). At 7 h postinfection, culture media were taken off andcells were rinsed with SLO buffer (25 mM HEPES [pH 7.4], 115 mM potassiumacetate, 2.5 mM Mg2Cl). They were then incubated for 15 min on ice with SLObuffer containing 1 mM DTT and 1 mg of SLO per ml and subsequently washedtwice with SLO buffer containing 1 mM DTT. To activate the SLO, the cells wereincubated for 30 min at 37°C with prewarmed SLO buffer containing 1 mM DTT.The cells were put on ice and directly rinsed with SLO wash buffer (50 mMHEPES [pH 7.4], 5 mM Mg2Cl, 2 mM EGTA, 50 mM KCl). This washing stepwas repeated once, and the cells were then washed with PBS and subjected tofixation with 3% paraformaldehyde (PFA) for 30 min. Proteinase K treatment ofSLO-permeabilized cells was carried out by preincubation of the cells after thePBS washing step for 5 min with proteinase K buffer, followed by incubation for30 min on ice with proteinase K buffer containing 50 mg of proteinase K per ml.Cells were then rinsed twice with proteinase K wash buffer (2 mM EGTA, 5 mMEDTA, 1.15 mM PMSF [pH 7.4]) and once with PBS containing 50 mM glycineand 1.15 mM PMSF, after which they were fixed with 3% PFA.

For classical indirect immunofluorescence, permeabilization of all cellularmembranes was performed after fixation with 3% PFA by treatment for 5 minwith 1% Triton X-100–5% FCS in PBS–50 mM glycine. The cells were washedthree times with PBS containing 50 mM glycine and 5% FCS.

Fluorescence labeling of the cells with antibodies was done by incubating themwith primary antibodies, followed by fluorescein isothiocyanate (FITC)- or tet-ramethyl rhodamine isocyanate (TRITC)-conjugated secondary antibodies. Theprimary antibodies used were rabbit anti-E (1:400), mouse anti-MN (undiluted),and rabbit anti-Mc (1:300) sera. The secondary antibodies used were FITC- orTRITC-conjugated goat anti-mouse and goat anti-rabbit immunoglobulin G(Cappel; 1:100).

Electron microscopy. Mouse L cells infected with MHV-A59 and BHK-21cells expressing the E protein were fixed at 5.30 and 4 h postinfection (hpi),respectively, and prepared for cryosectioning as described earlier (21). Thawedcryosections were double labeled with the E antibody at a dilution of 1:100 andwith Rab-1 antibodies (36) diluted 1:40. The double labeling was carried outaccording to the sequential protocol of Slot et al. (39). Embedding of cells inEpon was done as described elsewhere (13).

RESULTS

Kinetics of appearance of the E protein in MHV-A59-in-fected cells. Sera raised against purified MHV-A59 are verypoor in detecting the E protein. Thus, a fusion protein wasprepared in order to obtain protein E-specific antibodies forimmunological studies. It consisted of the 34-amino-acid car-boxy-terminal domain of MHV-A59 E protein preceded by theamino-terminal 161-residue ectodomain of the EAV GS pro-tein (Fig. 1B). By choosing GS as a fusion partner to enhanceimmune responses, we aimed to also acquire a polyclonal se-rum to this EAV protein. For purification purposes, the fusionprotein was extended amino terminally with a His-tag. Theprotein was produced in E. coli and injected into rabbits. Theimmune serum obtained was found to immunoprecipitate aprotein with an apparent molecular mass of 9.8 kDa fromlysates of radiolabeled cells infected with MHV-A59 or ex-pressing the viral E gene by using the vaccinia virus T7 system(data not shown). This molecular mass closely corresponds tothe predicted size of the E protein (9.6 kDa).

We used the antiserum to evaluate the synthesis of the Eprotein during MHV infection, comparing it with that of theother viral structural proteins. Cultures of infected cells weretherefore labeled for successive 1-h periods with 35S-labeledamino acids. Immunoprecipitates were prepared from the celllysates by using a polyclonal rabbit serum against MHV-A59that recognizes predominantly the N, M, and S proteins and byusing the rabbit serum against the E protein. Precipitates wereanalyzed in 15 and 20% PAG, respectively, as shown in Fig. 2A

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and B. For each of the viral proteins the time course of syn-thesis was determined by quantitation of the radioactivity inthe respective gel segments, the results of which, normalized toallow comparison, are compiled in Fig. 2C. It is clear that allfour proteins were synthesized with very similar kinetics. Theywere all first detected at between 3 and 4 hpi, after which theirproduction rapidly and simultaneously increased, reaching amaximal rate around 6 hpi, which was maintained for severalhours. Since the proteins were probably all immunoprecipi-tated with different efficiencies, these results do not allow anestimation of the relative molar ratios of their synthesis.

Stability of the E protein during infection. To further studythe fate of the E protein in infection, we performed a pulse-chase analysis, during which we again monitored the radioac-tivity in the E protein in comparison with that in the otherstructural proteins. In order to obtain an overall picture, weincluded in our analysis the fraction of the proteins that isreleased from the cells with virions. For this purpose we pre-pared total lysates in which cells and culture fluids were com-bined. Immunoprecipitates of M, S, and N proteins were pre-pared with the anti-MHV serum and analyzed in a 15% gel(Fig. 3A); E protein precipitated with the E-specific serum waselectrophoresed in a 20% gel (Fig. 3B). Quantitations of theradioactivity in the different proteins are graphically repre-sented in Fig. 3C. The data show that the structural proteinssynthesized at around 6 hpi turned over with quite similarkinetics. It was deduced from this and other experiments thatthe proteins had half-lives of approximately 4 h. A similarpattern of turnover was obtained when an otherwise identicalexperiment was performed in which protein synthesis wasblocked after the pulse-labeling by including cycloheximide(0.5 mM) in all chase media (data not shown). This result ruledout the possibility that the decline in the amount of radioactiveproteins was caused by a gradual shortage of antibodies duringthe immunoprecipitation as a result of the continued synthesisof (unlabeled) viral proteins.

Inspection of Fig. 3A reveals the appearance during thechase of a polypeptide (marked by an asterisk) running slightlyfaster in the gel than the normal set of M proteins. Thispolypeptide, which could also be observed in the experimentdepicted in Fig. 2, appeared concomitantly with the conversionof the unglycosylated M protein (M0) synthesized during thepulse into the different, slower-migrating O-glycosylated forms.Using domain-specific antibodies, we have demonstrated thatthe polypeptide represents an M protein that lacks its normalamino terminus (data not shown). Its discrete size, which isabout 2.4 kDa smaller than that of the M0 form, indicates thatit has completely lost the domain that is exposed at the luminalside of intracellular membranes, i.e., at the outside of virions.

Stability of individually expressed E protein. We also stud-ied the stability of the independently expressed E protein in apulse-chase experiment. Because no E protein appeared to bereleased into the culture medium (data not shown) only thecell-bound protein was analyzed. For comparison, we similarlyexpressed in parallel the M protein (known to be stable [34])and the EAV GS protein, which we found earlier to be subjectto degradation both in EAV-infected BHK-21 cells and whenexpressed individually in these cells (7). As the results com-piled in Fig. 4 show, the M and GS proteins behaved as ex-pected, although the latter protein, immunoprecipitated by theantiserum directed against GS-E fusion protein, appeared tobe less prone to turnover in the OST7-1 cells than in theBHK-21 cells used before. The stability of the E protein wascomparable with that of the GS protein under these conditions;its level remained constant initially but started to decreasethereafter.

Membrane integration of E occurs without signal sequencecleavage. The MHV-A59 E protein contains a rather longhydrophobic domain at its amino terminus which is likely tomediate its membrane integration. To find out whether itsmembrane insertion involves the functioning of a classical,cleavable signal sequence, we compared the size of the primarytranslation product with that of the membrane-integratedform. Capped RNA was transcribed in vitro from an E gene

FIG. 2. Kinetics of appearance of the E protein in MHV-A59-infected cells.MHV-infected and mock-infected OST7-1 cells were labeled with 35S-labeledamino acids (80 mCi/10-cm2 dish) for different 1-h periods starting at the indi-cated times after infection. Combined lysates of cells and culture media werethen prepared, and immunoprecipitations were carried out with different anti-sera. (A) Proteins precipitated with the polyclonal anti-MHV serum were ana-lyzed in 15% PAG. (B) Proteins precipitated by the anti-E (aE) or the preEserum were analyzed in 15% PAG. Radioactivities in the bands representing thedifferent viral proteins were quantitated, taking for M all the different forms,including the lower band indicated by an asterisk. The results are compiled inpanel C. They were normalized by placing the added total of all measurementsfor each protein at 100 and expressing each measurement as the fraction of thistotal.



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plasmid construct and translated in a reticulocyte lysate systemin the absence and in the presence of rough microsomal mem-branes. The labeled products were immunoprecipitated by us-ing the E antiserum, and their electrophoretic mobilities were

analyzed in parallel with that of E protein synthesized in trans-fected cells by using the vaccinia virus expression system. Fig-ure 5A shows that the E proteins synthesized in vitro and incells comigrated. Apparently, no signal sequence is cleavedduring or after membrane integration of the protein unless thesize reduction that would accompany such cleavage is compen-sated for by a secondary modification, for which we have noindications. The MHV-A59 E protein sequence does not con-tain an N-glycosylation motif. Moreover, as we show in Fig. 5B,we were unable to confirm the conclusion made by Yu et al.(49), on the basis of hydroxylamine treatment, that the Eprotein is acylated: in our experiment the protein remainedunaffected. Attempts to label the protein in infected cells with3H-labeled palmitic acid were equally unsuccessful (notshown). These observations are consistent with those of otherswho were similarly unable to label the TGEV E protein ininfected cells with 3H-labeled palmitic acid (12).

Topology of the membrane-assembled E protein. Besidesthe lack of a cleaved signal sequence at its amino terminus theE protein also lacks the typical anchor sequence occurring inthe carboxy-terminal region of so many other membrane pro-teins. Rather, its hydropathy plot suggests that the polypeptideis buried within the lipid bilayer for most of its amino-terminal60 residues. To determine the disposition of the residual more-hydrophilic carboxy terminus, we took an immunofluorescenceapproach based on the availability of an antiserum specificallyrecognizing this part of the protein. In this assay cells werepermeabilized selectively in their plasma membrane by usingSLO to allow intracellular access of antibodies. The assay wasfirst tested with cells expressing the M protein whose mem-brane topology has been well established (33, 35): its amino-and carboxy-terminal domains are exposed luminally and cy-

FIG. 3. Stability of the E protein in MHV-A59 infection. MHV-infected OST7-1 cells were labeled with 35S-labeled amino acids (80 mCi/10-cm2 dish) for 30 minstarting at 6 hpi. Combined lysates of cells and culture media were prepared either immediately or after different chase periods as indicated. Viral proteins wereimmunoprecipitated with polyclonal anti-MHV serum (aMHV) or anti-E (aE) serum and analyzed in 15% (A) and 20% (B) PAG, respectively. The radioactivitiesin the viral proteins were quantitated, including for S protein also the cleaved form of the protein and for M the faster-migrating band indicated by an asterisk (C).The results are compiled in panel C. For each protein the radioactivity in the chase samples was related to that observed after the pulse labeling, which was set at 100.

FIG. 4. Stability of the expressed E protein. vTF7-3-infected OST7-1 cellstransfected with pT7Ts-E, pTUM-M, or pAVI02 plasmids containing the E, theM, and the EAV GS genes, respectively, were labeled with 35S-labeled aminoacids (80 mCi/10-cm2 dish) for 1 h starting at 6 hpi. Cell lysates were preparedimmediately after the labeling or after various chase times as indicated, andimmunoprecipitations were carried out with the antisera anti-E (aE), pre-anti-E(preaE), and anti-MHV (aMHV). Proteins were analyzed in 20% PAG.



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toplasmically, respectively, and antibodies directed to thesedomains are available. Accordingly, surface-permeabilizedcells could not be stained with the anti-MN antibodies unlessthe intracellular membranes were also permeabilized by usingTriton X-100 (Fig. 6). In contrast, the anti-MC antibodiesreadily stained the surface-permeabilized cells, thus confirmingthe cytoplasmic exposure of the carboxy terminus. Consis-tently, staining was fully abrogated by prior treatment of thepermeabilized cells with proteinase K, a treatment that onlyaffected the cytoplasmically exposed protein domains since theamino terminus of the M protein remained intact and couldstill be visualized by the anti-MN antibodies after additionalTriton X-100 permeabilization. This latter treatment did notenable staining with the anti-MC antibodies, indicating that theM protein assumes one defined membrane topology, in con-trast to the TGEV M protein, which was reported to take twoalternative orientations (31).

When the immunofluorescence assay was applied to cellsexpressing the E protein, it became clear that its carboxy ter-minus protrudes into the cytoplasm. The domain was accessi-ble to the anti-E antibodies after surface permeabilization(Fig. 6). Proteinase K treatment completely abolished thestaining, and subsequent permeabilization with Triton X-100

did not reveal any “hidden” carboxy-terminal domains, a resultconsistent with a unique membrane topology.

A cytoplasmic exposure of the E protein’s carboxy terminuswould topologically correlate with a disposition of this domainon the inside of MHV particles. Accordingly, we were unableto label VLPs, obtained by expressing the MHV M and Eproteins, in an immunogold labeling with the anti-E antibodies,as judged under the electron microscope (data not shown). Tofurther corroborate this point, we also used a biochemicalapproach. Because of its great sensitivity, we performed animmunoisolation of radioactively labeled virus particles fromthe culture medium of MHV-A59-infected cells by using var-ious antibodies. As the protein pattern of Fig. 7A shows, theS-specific antibodies (Fig. 7A, aS) not only isolated S proteinbut in addition all other structural proteins M, N, and E, thelatter one being visible only after prolonged exposure (notshown). Similarly, viral particles could be isolated with theanti-MN antibodies but not with the anti-MC antibodies, afinding consistent with the known exterior and interior dispo-sition of the corresponding M domains, respectively. As ex-pected, no trace of viral particles was detected when the anti-Eserum was used in the immunoisolation, confirming the inte-rior disposition of the E protein’s carboxy terminus in virions.

Due to its very low abundance, the E protein is difficult todetected in MHV-A59 particles. We nevertheless analyzed theeffect of proteolytically removing the exposed domains of theviral membrane proteins. Figure 7B shows that treatment of35S-labeled virions eliminated the epitope present in the aminoterminus of the M protein recognized by the monoclonal J1.3(aMN). It also reduced the size of the polypeptide by about 2.5kDa as shown previously (33). The E protein seemed unaf-fected. Its appearance as a diffuse band, visible only after longexposure of the gel, did not change as a result of the treatment.The mere fact that the protein remained precipitable by theanti-E antibody implies that the carboxy terminus was indeedprotected. In addition, it shows that no other part(s) of theprotein is significantly exposed on the outside of virions.

Electron microscopic observations. One of the markedobservations made during the immunofluorescence studiesdescribed above was the peculiar punctate staining patternof the expressed E protein. To study this aspect in moredetail, BHK-21 cells expressing the protein were preparedfor and then analyzed by electron microscopy. It appearedthat the E protein induced the formation of characteristicelectron-dense structures (Fig. 8A) that were not seen incontrol cells, for instance, when the M protein was ex-pressed similarly. They were, however, also observed inMHV-A59-infected L cells (as shown after cryosectioning inFig. 8B) and are actually very reminiscent of the tubularstructures described in MHV-A59-infected cells many yearsago (4). They seem to consist of masses of tubular, smoothmembranes with much curvature that form complicated net-works. The membrane clusters are relatively homogeneousin that other organelles are excluded. Continuities with theendoplasmic reticulum (ER) are, however, often observed.The structures were obviously induced by the E proteinsince they labeled heavily with the anti-E antibody (Fig. 8Bto D). In fact, in highly expressing cells the E protein oftenappeared to localize almost exclusively to them. Interest-ingly, the tubular structures are part of the ER-intermediatecompartment (IC) network, as was demonstrated by theircolabeling on cryosections with a marker for these compart-ments, Rab-1 (Fig. 8B to D; see also references 14 and 36).

FIG. 5. E protein membrane integration without cleavage of a signal se-quence. (A) RNA transcribed in vitro from plasmid pT7Ts-E was translated in arabbit reticulocyte lysate in the presence or absence of canine microsomal mem-branes (MM). In parallel, vTF7-3-infected OST7-1 cells transfected withpT7Ts-E were labeled (80 mCi/10-cm2 dish) from 6 to 7 hpi and then lysed.Immunoprecipitations were carried out with the anti-E (aE) serum and the preEserum, and the proteins were analyzed in 20% PAG. (B) A similar OST7-1-derived immunoprecipitate of the E protein was split up into two parts, whichwere both incubated in 1 M Tris-Cl (pH 8.0), one of which also contained 1 Mhydroxylamine. Samples were then diluted with Laemmli sample buffer andanalyzed in 20% PAG. Untreated control immunoprecipitates prepared withanti-E and preE serum were included in the analysis for comparison. Note thatthe presence of hydroxylamine caused a broadening of the protein band in theright lane.



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DISCUSSION

The remarkable role of the E protein in coronavirus assem-bly prompted the detailed characterization of the envelopeprotein described here. E protein emerges from these studiesas a unique protein. The unglycosylated polypeptide appears inMHV-A59-infected cells synchronously with all the other viralstructural proteins. It is assembled in the ER membrane byvirtue of an uncleaved signal sequence assuming a complexmembrane structure which leaves its carboxy terminus in thecytoplasm. In virions this domain is oriented inwards, facingthe nucleocapsid. When expressed independently, the E pro-tein accumulates in and induces the coalescence of the mem-branes of the ER-IC, giving rise to characteristic structuresthat also appear in infected cells.

The E protein appears to integrate into the ER membranewithout modifications. Membrane insertion is mediated by asignal within the large hydrophobic domain, most likely by the28-residue stretch preceding the conserved lysine at position38. No cleavage of this domain is predicted nor was it observed.Hence, our biochemical analyses of permeabilized cells ex-pressing the protein and of viral particles lead to a picture of alargely membrane-embedded E protein, of which only the;23-residue hydrophilic tail is exposed cytoplasmically and onthe inside of virions. No part of the protein was detectablyexposed on the virion outside, the topological equivalent of theER lumen. The amino terminus may be oriented to either sideof the membrane. Other, more biophysical methods will berequired to determine the fine structure of the membrane-integral part of the molecule which is sufficiently long to span

the lipid bilayer twice. It will be interesting to find out to whichside of the membrane the conserved lysine (position 38) isoriented, what the disposition is of the conserved cysteinesimmediately downstream of this lysine, and what structuralposition the proline at position 54 takes that is absolutelyconserved among all coronavirus E proteins (10, 38).

In earlier immunofluorescence studies the E protein hasbeen detected at the surface of infected cells. With antibodiesprepared against the whole E protein, positive but weak stain-ing of unfixed cells infected with infectious bronchitis virus wasobserved (40). Surface staining was also reported for MHV-(49) and TGEV-infected cells, as well as for insect cells ex-pressing the TGEV E protein by a baculovirus vector (12). Theantibodies used in these studies were directed against the pro-tein’s carboxy terminus, leading Godet et al. (12) to the sug-gestion that this region of the molecule is translocated acrossthe membrane, a finding which is at variance with our obser-vations. The reasons for this discrepancy are unknown andremain to be elucidated.

In MHV-infected cells the structural proteins are synthe-sized from subgenomic mRNAs. These mRNAs are synthe-sized in different molar amounts but at constant relative ratios(17, 24). Except for the smallest mRNA, encoding just the Nprotein, they are all structurally polycistronic but express onlytheir 59-terminal open reading frame (ORF). The E protein,however, is translated from a downstream ORF by internalribosome entry (3, 44). Our quantitations of the synthesis ofthe S, N, M, and E proteins during infection show similarkinetics for all of them. Although the proteins are probably

FIG. 6. Location of the carboxy terminus of the membrane-integrated E protein. BHK-21 cells expressing the M protein or the E protein were permeabilized byusing either SLO or Triton X-100 and stained for immunofluorescence with the indicated antibodies. In some cases, the SLO-permeabilized cells were first treated withproteinase K (Prot.K) before the staining, while in other cases the SLO-permeabilized and proteinase K-treated cells were additionally permeabilized by using TritonX-100 and then incubated with the antibodies. aMC, peptide antiserum to the M protein’s C-terminal tail; aMN, monoclonal antibody J1.3 specific for the M protein’sN terminus.



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produced in very different molar amounts, their time courses ofsynthesis were indistinguishable.

In one of the first extensive electron microscopic studies ofcoronavirus-infected cells David-Ferreira and Manaker (4) de-

scribed the appearance of structures “formed by closely inter-woven, membrane-limited tubules.” These structures, whichwere termed tubular bodies, were induced by the MHV-A59infection. They appear to be very similar to what we found inour present studies, not only in infected cells but also in re-sponse to the independent expression of the E protein. Thisobservation thus links the induction of the structures directly tothe E protein. Moreover, their colabeling with antibodies to Eand to the ER-IC marker Rab-1 suggests that they are derivedby the coalescence of these pre-Golgi membranes. Although itsprecise cellular localization was beyond the scope of this study,our preliminary observations suggest that the expressed E pro-tein accumulates in membranes of the ER-IC. We and othershave shown earlier that budding of coronaviruses occurs atthese particular membranes (18, 21, 45, 46). The reasons forthis have so far remained obscure because neither of the majorviral envelope proteins was found to localize to these mem-branes (20, 29). If the E protein by itself indeed localizes topre-Golgi membranes, this would most likely provide the ex-planation. By its interactions with the M protein the E proteinmight retain M protein molecules in the early compartments,allowing the accumulation of the large lateral assemblies whereviral particles are formed (22, 29).

While numerous viruses have been shown to variously in-duce novel structures in infected cells, rarely have these struc-tures been assigned to the action of a particular viral protein.An interesting exception are the tubular networks of smoothmembranes that were reported by Hobman et al. (15, 16) toarise in cells upon expression of the rubella virus E1 glyco-protein. The characteristics of these structures are very similarto the ones described here, notably with respect to their mor-phology, their continuity with ER membranes, their labelingwith antibodies to ER-IC marker proteins, and their insensi-tivity to brefeldin A (data not shown). The E1-induced tubularnetworks were purified from cells by immunoisolation andshown to likely correspond to hypertrophied ER exit sites (16).

The convoluted appearance of the tubular membranes thatwe observed in response to the E protein may point to animportant biological feature of the protein in coronavirus mor-phogenesis. It suggests that the E protein has a tendency toinduce curvature into membranes. Earlier we hypothesizedthat this might be one of two possible roles that the proteinmight have during the budding of coronaviral particles (48).The alternative role was in the closing of the neck of thenascent particle, causing the pinching off of the virion. It is stilltoo early to decide whether the protein indeed serves one ofthese functions. Recent support for an important function ofthe E protein in virion morphogenesis comes from analyses ofMHV-A59 E gene mutants (10). Mutations introduced into theE protein’s hydrophilic tail by targeted RNA recombinationyielded viruses that were markedly thermolabile, suggestingthat there was a flaw in their structure. The particles of one ofthe viruses were viewed by electron microscopy and appearedto be aberrant and heterogeneous in their morphology. Insteadof the normal rounded structures, most virions had elongated,tubular forms, often pinched at multiple points, producingdumbbell-shaped structures. The pictures suggest that the Eprotein indeed is important for creating the membrane curva-ture needed to acquire the rounded, stable, and infectiousparticle phenotype.

The occurrence of small membrane proteins appears to bequite a general feature of enveloped RNA viruses. For some ofthese a function has been established such as for the ion-channel M2 protein of influenza virus (30) and for the alpha-virus 6K protein which is important in the final assembly andbudding of virions (25, 27). Interestingly, we recently discov-

FIG. 7. Topology of the E protein in MHV-A59 virions: the carboxy-terminaldomain is not exposed on the outside. (A) Immunoaffinity isolation of virions.OST7-1 cells grown in a 10-cm2 dish and infected with MHV-A59 were labeledwith 35S-labeled amino acids at 6 to 9 hpi, after which the cells were lysed andprocessed for immunoprecipitation with anti-MHV (aMHV) and anti-E (aE)(right panel). The culture medium containing released virus particles was clari-fied, and aliquots were first incubated with the antibodies as indicated (forexplanation, see previous figures; aS, monoclonal antibody J7.6 recognizing anepitope in the S ectodomain; C, control monoclonal antibody) and subsequentlywith Staphylococcus aureus bacteria to adsorb the antibodies and associated viralparticles, which were analyzed in 20% PAG. (B) Protease protection analysis.The culture medium of a similarly labeled 10-cm2 dish of infected cells was splitand either treated with proteinase K (Prot. K) or incubated without enzyme.Concentrated lysis buffer was then added, and standard immunoprecipitationswere carried out with the antibodies indicated (for explanations, see the legendsto previous figures). Proteins were analyzed in 20% PAG. From the upper panel,which was exposed for 4 h, the region containing the E protein is shown after alonger exposure (4 days) in the lower panel.



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ered that a small membrane protein also occurs in arterivi-ruses (41), which constitute another genus in the order ofNidovirales (8). This 67-residue E protein is a structuralcomponent of EAV. The polypeptide is very hydrophobic,with a basic carboxy terminus, and is membrane associatedin infected cells. Importantly, the protein appeared to beessential for the production of infectious viral particles. Inview of these findings it is surprising that no small mem-brane protein has so far been identified in toroviruses, the

second family of viruses belonging to the Coronaviridae.While these viruses do have an S protein and an M proteinvery similar to those of coronaviruses, no E protein homo-logue seems to be encoded. This suggests that the buddingof these viruses occurs differently from coronaviruses or thatthe function of the E protein is expressed otherwise. Weexpect that our continued comparative studies of these dif-ferent nidoviruses will eventually provide more detailed in-sight into the various ways these viruses assemble their particles.

FIG. 8. Electron microscopic analysis. (A) Epon section of BHK-21 cell expressing the E protein and fixed at 4 h posttransfection. The E protein induces theformation of electron-dense membrane structures that are often continuous with the rough ER (arrowheads). (B to D) The same structures in thawed cryosectionsdouble labeled for E (5-nm gold; arrows) and Rab-1 (10-nm gold; arrowheads). Panels: B, an MHV-infected cell fixed at 5.30 hpi, C and D, BHK-21 cells expressingthe E protein. Bars, 100 nm.



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ACKNOWLEDGMENT

This work was financially supported in part by Intervet InternationalB.V., which we gratefully acknowledge.

REFERENCES

1. Abraham, S., T. E. Kienzle, W. E. Lapps, and D. A. Brian. 1990. Sequenceand expression analysis of potential nonstructural proteins of 4.9, 4.8, 12.7,and 9.5 kDa encoded between the spike and membrane protein genes of thebovine coronavirus. Virology 177:488–495.

1a.Baudoux, P., C. Carrat, L. Besnardeau, B. Charley, and H. Laude. 1998.Coronavirus pseudoparticles formed with recombinant M and E proteinsinduce alpha interferon synthesis by leukocytes. J. Virol. 72:8636–8643.

2. Bredenbeek, P. 1990. Nucleic acid domains and proteins involved in thereplication of coronaviruses. Ph.D. thesis. Utrecht University, Utrecht, TheNetherlands.

3. Budzilowicz, C. J., and S. R. Weiss. 1987. In vitro synthesis of two polypep-tides from a nonstructural gene of coronavirus mouse hepatitis virus strainA59. Virology 159:509–515.

4. David-Ferreira, J. F., and R. A. Manaker. 1965. An electron microscopestudy of the development of a mouse hepatitis virus in tissue culture cells.J. Cell Biol. 24:57–78.

5. de Haan, C. A. M., P. Roestenberg, M. de Wit, A. A. F. de Vries, T. Nilsson,H. Vennema, and P. J. M. Rottier. 1998. Structural requirements for O-glycosylation of the mouse hepatitis virus membrane protein. J. Biol. Chem.273:29905–29914.

6. de Vries, A. A. F., E. D. Chirnside, M. C. Horzinek, and P. J. M. Rottier.1992. Structural proteins of equine arteritis virus. J. Virol. 66:6294–6303.

7. de Vries, A. A. F., M. J. B. Raamsman, H. A. van Dijk, M. C. Horzinek, andP. J. M. Rottier. 1995. The small envelope glycoprotein (Gs) of equinearteritis virus folds into three distinct monomers and a disulfide-linkeddimer. J. Virol. 69:3441–3448.

8. de Vries, A. A. F., M. C. Horzinek, P. J. M. Rottier, and R. J. de Groot. 1997.The genome organization of the Nidovirales: similarities and differencesbetween arteri-, toro-, and coronaviruses. Semin. Virol. 8:33–47.

9. Elroy-Stein, O., and B. Moss. 1990. Cytoplasmic expression system based onconstitutive synthesis of bacteriophage T7 RNA polymerase in mammaliancells. Proc. Natl. Acad. Sci. USA 87:6743–6747.

10. Fischer, F., C. F. Stegen, P. S. Masters, and W. A. Samsonoff. 1998. Analysisof constructed E gene mutants of mouse hepatitis virus confirms a pivotalrole for E protein in coronavirus assembly. J. Virol. 72:7885–7894.

11. Fuerst, T. R., E. G. Niles, F. W. Studier, and B. Moss. 1986. Eukaryotictransient-expression system based on recombinant vaccinia virus that syn-thesize bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA83:8122–8126.

12. Godet, M., R. L. Haridon, J.-F. Vautherot, and H. Laude. 1992. TGEVcoronavirus ORF4 encodes a membrane protein that is incorporated intovirions. Virology 188:666–675.

13. Griffiths, G. 1993. Fine structure immunocytochemistry. Springer-Verlag,Heidelberg, Germany.

14. Griffiths, G., M. Ericsson, J. Krijnse Locker, T. Nilsson, H. D. Soeling, B. L.Tang, S. H. Wong, and W. Hong. 1994. Ultrastructural localization of themammalian KDEL receptor in cultured cells and tissues. J. Cell Biol. 127:1557–1574.

15. Hobman, T. C., L. Woodward, and M. G. Farquhar. 1992. The rubella virusE1 glycoprotein is arrested in a novel post-ER, pre-Golgi compartment.J. Cell Biol. 18:795–811.

16. Hobman, T. C., B. Zhao, H. Chan, and M. G. Farquhar. 1998. Immunoiso-lation and characterization of a subdomain of the endoplasmic reticulumthat concentrates proteins involved in COPII vesicle biogenesis. Mol. Biol.Cell 9:1265–1278.

17. Jacobs, L., W. J. M. Spaan, M. C. Horzinek, and B. A. M. van der Zeijst.1981. Synthesis of subgenomic mRNAs of mouse hepatitis virus is initiatedindependently: evidence from UV transcription mapping. J. Virol. 39:401–412.

18. Klumperman, J., J. Krijnse Locker, A. Meijer, M. C. Horzinek, H. J. Geuze,and P. J. M. Rottier. 1994. Coronavirus M proteins accumulate in the Golgicomplex beyond the site of virion budding. J. Virol. 68:6523–6534.

19. Krijnse Locker, J., J. K. Rose, M. C. Horzinek, and P. J. M. Rottier. 1992.Membrane assembly of the triple-spanning coronavirus M protein: individualtransmembrane domains show preferred orientation. J. Biol. Chem. 267:21911–21918.

20. Krijnse Locker, J., G. Griffiths, M. C. Horzinek, and P. J. M. Rottier. 1992.O-Glycosylation of the coronavirus M protein: differential localization ofsialyltransferases in N- and O-glycosylation. J. Biol. Chem. 267:14094–14101.

21. Krijnse Locker, J., M. Ericsson, P. J. M. Rottier, and G. Griffiths. 1994.Characterization of the budding compartment of mouse hepatitis virus: ev-idence that transport from the RER to the Golgi complex requires only onevesicular transport step. J. Cell Biol. 124:55–70.

22. Krijnse Locker, J., D.-J.E. Opstelten, M. Ericsson, M. C. Horzinek, andP. J. M. Rottier. 1995. Oligomerization of a trans-Golgi/trans-Golgi network

retained protein occurs in the Golgi complex and may be part of its reten-tion. J. Biol. Chem. 270:8815–8821.

23. Kyte, J., and R. F. Doolittle. 1982. A simple method for displaying thehydropathic character of a protein. J. Mol. Biol. 157:105–132.

24. Leibowitz, J. L., K. C. Wilhelmsen, and C. W. Bond. 1981. The virus-specificintracellular RNA species of two murine coronaviruses: MHV-A59 andMHV-JHM. Virology 114:39–51.

25. Liljestrom, P., S. Lusa, D. Huylebroeck, and H. Garoff. 1991. In vitro mu-tagenesis of a full-length cDNA clone of Semliki Forest virus: the 6,000-molecular-weight membrane protein modulates virus release. J. Virol. 65:4107–4113.

26. Liu, D. X., and S. C. Inglis. 1991. Association of the infectious bronchitisvirus 3c protein with the virion envelope. Virology 185:911–917.

27. Loewy, A., J. Smyth, C.-H. von Bonsdorff, P. Liljestrom, and M. J.Schlesinger. 1995. The 6-kilodalton membrane protein of Semliki Forestvirus is involved in the budding process. J. Virol. 69:469–475.

28. Opstelten, D.-J.E., P. de Groote, M. C. Horzinek, H. Vennema, and P. J. M.Rottier. 1993. Disulfide bonds in folding and transport of the mouse hepatitisvirus glycoproteins, J. Virol. 67:7394–7401.

29. Opstelten, D.-J.E., M. J. B. Raamsman, K. Wolfs, M. C. Horzinek, andP. J. M. Rottier. 1995. Envelope glycoprotein interactions in coronavirusassembly. J. Cell Biol. 131:339–349.

30. Pinto, L. H., L. J. Holsinger, and R. A. Lamb. 1992. Influenza virus M2

protein has ion channel activity. Cell 69:517–528.31. Risco, C., I. M. Anton, C. Sune, A. M. Pedregosa, J. M. Martin-Alonso, F.

Parra, J. L. Carrascosa, and L. Enjuanes. 1995. Membrane protein mole-cules of transmissible gastroenteritis virus also expose the carboxy-terminalregion on the external surface of the virion. J. Virol. 69:5269–5277.

32. Rottier, P. J. M., W. J. M. Spaan, M. C. Horzinek, and B. A. M. van derZeijst. 1981. Translation of three mouse hepatitis virus strain A59 sub-genomic RNAs in Xenopus laevis oocytes. J. Virol. 38:20–26.

33. Rottier, P. J. M., D. Brandenburg, J. Armstrong, B. A. M. van der Zeijst, andG. Warren. 1984. Assembly in vitro of a spanning membrane protein of theendoplasmic reticulum: the E1 glycoprotein of coronavirus MHV-A59. Proc.Natl. Acad. Sci. USA 81:1421–1425.

34. Rottier, P. J. M. 1995. The coronavirus membrane protein, p. 115–139. InS. G. Siddell (ed.), The Coronaviridae. Plenum Press, New York, N.Y.

35. Rottier, P. J. M., G. W. Welling, S. Welling-Wester, H. G. M. Niesters, J. A.Lenstra, and B. A. M. van der Zeijst. 1986. Predicted membrane topology ofthe coronavirus protein E1. Biochemistry 25:1335–1339.

36. Saraste, J., U. Lahtinen, and B. Goud. 1995. Localization of the smallGTP-binding protein rab1p to early compartments of the secondary pathway.J. Cell Sci. 108:1541–1552.

37. Siddell, S. G. 1995. The Coronaviridae. Plenum Press, New York, N.Y.38. Siddell, S. G. 1995. The small-membrane protein, p. 181–189. In S. G. Siddell

(ed.), The Coronaviridae. Plenum Press, New York, N.Y.39. Slot, J. W., H. G. Geuze, S. Gigengack, G. E. Lienhard, and D. E. James.

1991. Immuno-localization of the insulin regulatable glucose transporter inbrown adipose tissue of the rat. J. Cell Biol. 113:123–135.

40. Smith, A. R., M. E. G. Boursnell, M. M. Binns, T. D. K. Brown, and S. C.Inglis. 1990. Identification of a new membrane-associated polypeptide spec-ified by the coronavirus infectious bronchitis virus. J. Gen. Virol. 71:3–11.

41. Snijder, E. J., H. van Tol, K. W. Pedersen, M. J. B. Raamsman, and A. A. F.de Vries. 1999. Identification of a novel structural protein of arteriviruses.J. Virol. 73:6335–6345.

42. Spaan, W. J. M., P. J. M. Rottier, M. C. Horzinek, and B. A. M. van derZeijst. 1981. Isolation and identification of virus specific mRNAs in cellsinfected with mouse hepatitis virus (MHV-A59). Virology 108:424–434.

43. Sutter, G., M. Ohlman, and V. Erfle. 1995. Non-replicating vaccinia virusvector efficiently expresses bacteriophage T7 RNA polymerase. FEBS Lett.371:9–12.

44. Thiel, V., and S. G. Siddell. 1994. Internal ribosome entry in the codingregion of murine hepatitis virus mRNA5. J. Gen. Virol. 75:3041–3046.

45. Tooze, J., S. A. Tooze, and G. Warren. 1984. Replication of coronavirusMHV-A59 in Sac2 cells: determination of the first site of budding of progenyvirions. Eur. J. Cell Biol. 33:281–293.

46. Tooze, S. A., J. Tooze, and G. Warren. 1988. Site of addition of N-acetyl-galactosamine to the E1 glycoprotein of mouse hepatitis virus-A59. J. CellBiol. 106:1475–1487.

47. Tung, F. Y. T., S. Abraham, M. Sethna, S. L. Hung, P. Sethna, B. G. Hogue,and D. A. Brian. 1992. The 9-kDa hydrophobic protein encoded at the 39 endof the porcine transmissible gastroenteritis coronavirus genome is mem-brane-associated. Virology 186:676–683.

48. Vennema, H., G.-J. Godeke, J. W. A. Rossen, W. F. Voorhout, M. C. Horzinek,D.-J.E. Opstelten, and P. J. M. Rottier. 1996. Nucleocapsid-independent assem-bly of coronavirus-like particles by coexpression of viral envelope proteins.EMBO J. 15:2020–2028.

49. Yu, X., W. Bi, S. R. Weiss, and J. L. Leibowitz. 1994. Mouse hepatitis virusgene 5b protein is a new virion envelope protein. Virology 202:1018–1023.



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2000 characterization of the coronavirus mouse hepatitis virus strain a59 small membrane protein e

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